DESCRIPTION: Highly Active Antiretroviral Therapies (HAART) have been successful at controlling viral load in HIV-infected patients and have significantly decreased the morbidity and mortality associated with HIV-1 infection. However, drug resistant HIV-1 strains arise and are transmitted between individuals, reducing the efficacy of currently available antivirals. Thus, identification of novel antiretrovirals (ARTs) that act by new mechanisms and have no cross-resistance with existing therapeutics is required. HIV-1 capsid (CA) is a viral protein essential for early and late events of the replication cycle and so far has been an untapped target, thus providing excellent opportunities for the discovery of novel ARTs that act by novel mechanisms of action. We screened an in-house chemical library of compounds and identified18E8, a small molecule that interferes with multimerization of HIV-1 CA. We demonstrated in cell-based assays with fully-infectious HIV that 18E8 showed broad antiretroviral activity (EC50 as low as ~1?M) against multiple laboratory strains and several multi-drug resistant clinical isolates. In additional preliminary experiments we showed that 18E8 exerts its antiretroviral activity by binding to HIV-1 CA. Six additional hits from this screening have not yet been characterized. In order to gain insight into the mode of action of 18E8 and to determine the specific step of the viral replication cycle that it affects, we performed time-of-drug-addition experiments that define how long the addition of 18E8 could be postponed before losing its antiviral activity in cell culture. Surprisingly, our preliminary data suggest that 18E8 targets an early step in the HIV replication cycle, after reverse transcription. 18E8 did not appear to affect late stages of the vial life cycle. Based on these data, we hypothesize that the unique mechanism of action of 18E8 is due to its binding in a manner different than other CA-targeting compounds that affect other steps of the virus life cycle. We will explore this hypothesis and use the same approaches to evaluate and characterize additional compound hits in the following two aims: Specific Aim 1. Functional and mechanistic characterization of 18E8 and other compounds Specific Aim 2. Crystallographic characterization of CA interactions with 18E8 and other compounds. The deliverables of the proposed studies include at least one novel CA-targeting antiviral with a new mechanism of action, its biological and structural mechanism of action that could guide the design of novel antivirals.